JP2009137973A - Use of dipeptidyl peptidase IV effectors for lowering blood glucose levels in mammals - Google Patents

Abstract

The present invention relates to an effector of dipeptidyl peptidase (DPIV) or DPIV-like enzyme activity intended to reduce hyperglycemia levels as seen in mammals in clinically inappropriate postprandial postprandial hyperglycemia. Providing methods using utilities.
Dipeptidyl peptidase (DPIV) or DPIV-like for lowering hyperglycemia levels as found in mammals with clinically inappropriate basal and post-prandeial hyperglycemia Use of enzyme activity effectors.
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Description

  In the present invention, an activity inhibitory effector (substrate, pseudo-substrate, inhibitor, binding protein, antibody, and the like) of an enzyme group having an activity similar to or the same as the enzyme activity of the enzyme dipeptidyl peptidase IV is administered. The present invention relates to a novel method for reducing the concentration.

  In addition to the proteolytic enzymes included in nonspecific proteolysis, proteolytic enzymes that cause specific degradation of proteins associated with endogenous peptide function control (activation, deactivation or regulation) are known [KIRSCHKE , H., LANGNER, J., RIEMANN, S., WIEDERANDERS, B., ANSORGE.S.and BOHLEY, P.Lysosomal cysteine proteases.Excerpta Medica (Ciba Foundation Symposium 75), 15 (1980); KRAUSSLICH, HGand WIMMER, E., Viral Proteinases. Ann. Rev. Biochem. 57, 701 (1987)].

  Such invertases, signal peptidases or enkephalinases have been discovered in the immune system as a result of neuropeptide research [GOMEZ, S., GLUSCHANKOF, P., LEPAGE, A., MARRAKCHI, N. and COHEN, P. Proc. Natl. Acad. Sci. USA 85, 5468 (1988); ANSORGE, S. and SCHoN, E., Histochem. 82, 41 (1987)].

  The amino acid proline, which is abundant in many peptide hormones, determines the structural properties of the peptide, so it is argued that proline-specific peptidases have a similar function to signal peptidases in the control of bioactive peptides [YARON, A., The Role of Proline in the Proteolytic Regulation of Biologically Active Peptides.Biopolymers 26,215 (1987): WALTER, R., SIMMONS, WHand YOSHIMOTO, T., Proline Specific Endo-and Exopeptidases.Mol.Cell. Biochem. 30,111 (1980); VANHOOF G., GOOSSENS, F., DE MEESTER, I., HENDRIKS, D. and SCHARPE'S., Proline motifs and their biological processing. FASEB Journal 9, 736 (1995)]. Since proline has an exceptional structure, it gives both such conformation and stability to avoid such degradation by non-specific proteolytic enzymes [KESSLER, H., Conformation and biological activity. .Angew.Chem.94,509 (1982)]. On the other hand, enzymes that act very specifically on sequences containing proline (including HIV protease, cyclophilin, etc.) are important targets in medicinal chemistry. In particular, peptidase activities that cleave downstream of proline, such as proline endopeptidase (PEP) and dipeptidyl peptidase IV (DPIV), have been linked to selective degradation by enzymes and regulation of the physiological activity of natural peptide substrates. PEP has been shown to be involved in memory and learning activities, and DPIV has been shown to be involved in information transmission in the immune response [ISHIURA, S., TSUKAHARA.T., TABIRA., SHIMIZU.T., ARAHATA K. and SUGITA.H., FEBS-Letters 260.131 (1990); HEGEN.M., NIEDOBITEK.G., KLEIN, CE, STEIN, H. and FLEISCHER, B., J. of Immunology 144, 2908 (1990)].

  In addition to high proline specificity, these enzymes can selectively recognize and cleave peptide bonds containing the amino acid alanine contained in typical substrates. At present, it is under debate whether an alanine-containing peptide takes a similar conformation to a structurally correlated proline-containing peptide. Recently, such characteristics were described in a point mutation experiment in which proline and alanine in protein were exchanged [DODGE.RWand SCHERAGA, HA, Folding and unfolding kinetics of the proline-to-alanine mutants of bovine pancreatic ribonuclease A. Biochemistry 35 (5) 1548 (1996)].

  DPIV or DPIV-like activity present in blood (ie, cytosolic DPII has almost the same substrate specificity as DPIV) is very specific, with proline or alanine in the second position from the N-terminus The dipeptide is released from the N-terminus of the bioactive peptide. From the above, it was concluded that this enzyme is involved in the regulation of polypeptide activity in the body [VANHOOF, G., GOOSSENS, F., DE MEESTER, I., HENDRIKS, D., and SCHARPE, S., Proline motifs and their biological processing, FASEB Journal 9,736 (1995)].

Glucose-dependent insulinotropic polypeptides: gastric inhibitory polypeptides 1-42 (GIP _1-42 ) and glucagon-like peptide amide-17-36 (GLP-1 _7-36 ) are insulin induced by glucose from the pancreas A hormone that increases secretion (incretin) and a substrate for DPIV. This enzyme releases dipeptides tyrosylalanine and histidylalanine from the N-terminal of these peptides in vivo and in vitro, respectively [MENTLEIN, R., GALLWITZ, B., and SCHMIDT, WE, Dipeptidyl Peptidase IV hydrolyzes gastric inhibitory polypeptide, glucagon-like peptide-1 (7-36) amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur. J. Biochem. 214, 829 (1993)].

  Reduction of substrate degradation due to DPIV or DPIV-like enzyme activity in the body can contribute to effective suppression of undesirable enzyme activity, both under laboratory conditions and under pathological conditions in mammals [ DEMUTH, H.-U., Recent developments in the irreversible inhibition of serine and cysteine proteases.J.Enzyme Inhibition 3,249-278 (1990); DEMUTH, H.-U.and HEINS, J., On the catalytic Mechanism of Dipeptidyl Peptidase IV. In Dipeptidyl Peptidase IV (CD 26) in Metabolism and the Immune Response (B. Fleicher, Ed.) RGLandes, Biomedical Publishers, Georgetown, 1-35 (1995)]. In short, non-insulin dependent diabetes mellitus is associated with insulin resistance and insulin secretion, which is inadequate for normal glucose levels and is a proteolytic enzyme May be associated with more than the blood incretin levels involved [BROWN, JC, DAHL, M., KWAWK, S., MCINTOSH, CHS, OTTE, SCand PEDERSON, RAPeptides 2,241 (1981) ; SCHMIDT, WE, SIEGEL, EG, GALLWITZ, B.KUMMEL, H., EBERT, R.and CREUTZFELDT, W., Characterization of the insulinotropic activity of fragments derived from gastric inhibitory polypeptide.Diabetologia 29,591A (1986); ADELHORST, K., HEDEGAARD, BB, KNUDSEN, LBand KIRK, O., Structure-activity studies of glucagon-like peptide. J. Biol. Chem. 296, 6275 (1994)].

  Currently, insulin dependent diabetes mellitus (IDDM) is treated and treated in various forms of insulin (isolated from calf fetus or porcine pancreas or produced as a recombinant molecule) to patients. ing. Non-insulin dependent diabetes mellitus (NIDDM) has been treated by stimulating insulin secretion by administering diet or sulfonylurea or by using with biguanides to increase glucose intake. Like modern treatment methods, traditional treatment methods of IDDM are characterized by significant patient efforts, high medical costs, and a significant reduction in patient quality of life. Standard therapies (daily intravenous injections of insulin) have been practiced since the 1930s and have been instructed to treat acute symptoms, but long-term administration results in vascular disease and nerve damage [LACY , P., Status of Islet Cell Transplantation, Diabetes Care 16 (3) 76 (1993)]. Recent methods such as subcutaneous infusions of depot-implants (requiring insulin that does not require daily injections, low doses, and no proteolytic action) have been tried as well as complete transplantation (or transplantation) of Langerhans Island It is in. However, such transplants are expensive. In addition, there are dangerous surgical procedures, and in the case of transplantation, it is necessary to avoid immunosuppression or immune reaction [LACY, P., Treating Diabetes with Transplanted Cells. Sci. Americ. 273 (1) 40- 46 (1995)]. Attempts to remove reduced glucose have not been successful. In the case of NIDDM, many patients who are treated with sulfonylureas to stimulate the secretion of endogenous insulin become resistant to these drugs. Furthermore, there are few successful cases of increasing glucose removal using biguanides.

  In contrast to the above therapeutic methods, the administration of the highly effective small molecule enzyme inhibitors proposed here is a cost-effective alternative. Such inhibitors for various proteolytic enzymes are already used as antihypertensive agents, immunosuppressive agents and antiviral agents. Chemical design of molecules with consideration for stability, transport, and cleaning value properties has changed their efficacy and have even adapted compounds to individual differences between organs [SANDLER.M. And SMITH , HJ, eds., Design of Enzyme Inhibitors as Drugs. Oxford University Press, Oxford (1989); MUNROE, JE, SHEPHERD, TA, JUNGHEIM, LN, HORNBACK, WJ, HATCH, SD, MUESING, MA, WISKERCHEN, MA, SU, KS, CAMPANALE, KM, BAXTER, AJ, and COLACINO, JM, Potent, orally biovailable HIV-1 protease inhibitors containing noncoded D-amino acids. Bioorg. Medicinal Chem. Letters 5 (23) 2897 (1995)].

Gastric inhibitory polypeptide 1 which is an endogenous insulinotropic peptide (or administered from outside), which induces suppression of dipeptidyl peptidase (DPIV or CD26) or DPIV-like enzyme activity in mammalian blood by an enzyme effector -42 (GIP _1-42 ) and glucagon-like peptide amide-1 7-36 (GLP-1 _7-36 ) (or analogs of these peptides), a new and simple method that lowers blood glucose levels This method is the aim of the present invention. Normally, it is degraded to DPIV and DPIV-like enzymes, and the concentration of these peptides or analogs thereof is lowered, but this is expected to be suppressed or delayed.

  The present invention is based on the discovery that glucose tolerance is improved by inhibiting the enzyme activity or DPIV-like enzyme activity of dipeptidyl peptidase (DPIV or CD26) in mammalian blood.

The present inventor observed the following.
1. Incretin (or analogs thereof) that are stimulated by glucose and endogenously released or externally administered by suppressing the enzyme activity or DPIV-like enzyme activity of dipeptidyl peptidase (DPIV or CD26) To increase the stability of. That is, administering an effector of DPIV or DPIV-like protein can be used to control the degradation of incretin in blood.
2. Increased biological stability of incretin (or their analogs) alters the insulin response.
3. Decrease in dipeptidyl peptidase (DPIV or CD26) or DPIV-like enzyme increases the stability of incretin in the blood, followed by a change in glucose removal by insulin. That is, it shows that the glucose tolerance function can be improved by administration of the DPIV effector.
Thus, the present invention is directed to dipeptidyl peptidase (DPIV) or DPIV-like enzyme activity aimed at lowering hyperglycemia levels as seen in mammals with clinically inappropriate postprandial postprandial hyperglycemia. It relates to the effect of the effector. A feature of the present invention is that administration of an effector of DPIV or DPIV-like enzyme activity prevents or delays pathological dysfunction of mammalian metabolic systems such as diabetes, hyperlipidemia, metabolic acidosis and diabetes mellitus. It is to let you.

  In more preferred terms, the present invention demonstrates clinically inappropriate basal postprandial hyperglycemia by administering a therapeutically effective amount of an effector of dipeptidyl peptidase (DPIV) or DPIV-like enzyme activity. It relates to a method of lowering hyperglycemia levels as found in some mammals.

  In other words, the present invention relates to the use of dipeptidyl peptidase (DPIV) or dipeptidyl peptidase (DPIV) for use in lowering hyperglycemia levels as found in mammals exhibiting clinically inappropriate post-prandial hyperglycemia. It relates to an effector of DPIV-like enzyme activity.

  According to the present invention, the administered effector of DPIV and DPIV-like enzymes is a pharmaceutical formulation such as an enzyme inhibitor, a substrate, a pseudo-substrate, a DPIV gene expression inhibitor, a binding protein or an antibody of a target enzyme protein, or a mammal. The pharmaceutical formulation is a combination with another compound that reduces the DPIV and DPIV-like protein concentration or enzyme activity. The effector referred to in the present invention is, for example, a dipeptide derivative such as alanyl pyrrolidide or isoleucyl thiazolidide, or a simulated dipeptidide, or a DPIV inhibitor such as the pseudosubstrate N-valylbrolyl-O-benzoylhydroxylamine. Such compounds are described in the literature [DEMUTH, H.-U., Recent developments in the irreversible inhibition of serine and cysteine proteases. J. Enzyme Inhibition 3,249 (1990)], or described in the literature. Synthesized according to the method.

  The method referred to in the present invention is a novel method for reducing a high blood glucose concentration in mammalian blood.

  The present invention is simple and commercially useful and suitable for the treatment of human diseases caused by inappropriate hyperglycemia.

  The effector is administered in the form of a formulation combined with a state-of-the-art material for drug delivery. Effectors are administered either parenterally (intravenous administration in saline) or enterally (orally, formulated with common antibody substances).

  Due to the internal stability and bioavailability of the effector, normalization of the blood glucose concentration expected with single or multiple doses is reached. The dose of such effectors varies from 1.0 mg to 10.0 mg per kilo, for example in the case of aminoacyl thiazolidides as DPIV inhibitors.

EXAMPLES Example 1: Inhibition of incretin GIP _1-42 and GLP-1 _7-36 hydrolysis catalyzed by DPIV in vivo In vitro, DPIV and DPIV-like enzyme activity using purified enzyme or human serum It is possible to suppress hydrolysis due to (FIG. 1).

According to the present invention, a 20% serum fraction containing 30 mM GIP _1-42 or 30 mM GLP-1 _7-36 and a reversible DPIV inhibitor 20 mM isoleucyl thiazolidide (1a) is added to pH 7.6, 30 By culturing in a test tube at 24 ° C. for 24 hours, hydrolysis of both peptide hormones catalyzed by the enzyme is completely suppressed (1b and 1c, both in the upper spectrum). Synthetic GIP _1-42 (5 mM) and synthetic GLP-1 _7-36 (15 μM) were cultured in 0.1 mM Tricine buffer containing human serum (20%) at pH 7.6 and 30 ° C. for 24 hours. A sample for culture assay (2.5 pmol in the case of GIP _1-42 and 7.5 pmol in the case of GLP-1 _7-36 ) was extracted over time. Samples were co-crystallized with 2 ', 6'-dihydroxyacetophenone and analyzed by MALDI-TOF mass spectrometry. The spectrum (FIG. 1) shows the cumulative value of 250 single laser shots per sample.

(1b) The signal at m / z 4980 ± 5.3 is GIP _1-42 (molecular weight 4975.6), a substrate for DPIV, and the signal at m / z 4745.2 ± 5.5 is the product GIP released from DPIV. _3-42 (molecular weight 4740.4).

(1c) The signal of m / z 3325.0 ± 1.2 is GLP _7-36 (molecular weight 3297.7) which is a substrate of DPIV, and the signal of m / z 3116.7 ± 1.3 is released from DPIV. Product GLP-1 _9-36 (molecular weight 3089.6).

  In a control experiment with no inhibitor, incretin was almost completely digested. (Figures 1b and 1c, both lower spectra)

Example 2: Inhibition of degradation of GLP-1 _7-36 by the DPIV inhibitor isoleucyl thiazolidide in vivo The DPIV inhibitor isoleucyl thiazolidide in rats circulatory system (0 containing 1.5M inhibitor) Metabolic analysis of natural incretin (in this case GLP-1 _7-36 ) in the presence or absence of 9% saline intravenous) and in control experiments.

When the in vivo concentration of the inhibitor isoleucyl thiazolidide in the test animal (n = 5) was 0.1 mg / kg, the insulinotropic peptide hormone GLP-1 _7-36 was not degraded during the experiment (FIG. 2). . To analyze incretin metabolites in the presence or presence of DPIV inhibitors, test and control animals were further treated with 50-100 pM of ¹²⁵ I-GLP-1 _7-36 (specific activity, approximately 1 [Ci / pM] (20 minutes after intravenous injection of the first inhibitor and / or salt). Blood samples were collected after 2-5 minutes incubation and plasma was extracted with 20% acetonitrile. Thereafter, the peptide extract was separated by reverse phase high performance liquid chromatography. The fraction eluting at 12-18 minutes was collected and the radioactivity was measured. The data was described as a value relative to the maximum value in counts per minute [cpm].

Example 3: Change in insulin response and reduction of blood glucose level following intravenous administration of the DPIV inhibitor isoleucyl thiazolidide in vivo The figure shows the presence of isoleucyl thiazolidide (0.1 mg / kg) or Figure 2 shows blood glucose and insulin responses to glucose administration into the rat duodenum under deficiency. In the group of animals administered with the DPIV effector, the blood glucose concentration decreased sharply compared to the control group. The effect observed here is volume dependent and is reversible even after the infusion is terminated with 0.05 mg / min of the DPIV inhibitor isoleucyl thiazolidide per kilogram of rat. No effect was seen in the inhibitor-treated control animals in which an equal amount of glucose was intravenously administered to animals in which the duodenum was stimulated with glucose. FIG. 3 shows the above relationship. This indicates an inhibitor-dependent change in plasma parameters. A: DPIV activity, B: plasma insulin level, C: blood glucose level A test animal (n = 5, male Wester rat, 200-225 g) is first prepared with 0.9% sodium chloride containing 1.5 M isoleucil thiazolidide. Water (▲) or an equal amount of 0.9% saline (■) was administered (control group n = 5). The test group was further infused with 0.75 M inhibitor per minute for 30 minutes (*), and the control group was infused with 0.9% saline containing no inhibitor for the same time. At start time t = 0, all animals received 1 g / kg of 40% (w / v) glucose solution into the duodenum. Blood was collected from all test animals every 10 minutes. Whole blood was used to analyze glucose (Lifescan One Touch II analyzer), and DPIV activity and insulin concentration were analyzed using plasma. Insulin-labeled immunoassay was sensitive in the range of 10-160 mU / ml [REDERSON, RA, BUCHAN, AMJ, ZAHEDI-ASH, S, .CHEN, CB & BROWN, JCReg. Peptides. 3, 53-63 (1982 ]]. DPIV activity was evaluated spectrophotometrically [DEMUTH, H.-U. and HEINS, J., On the catalytic Mechanism of Dipeptidyl Peptidase IV. In Dipeptidyl Peptidase IV (CD 26) in Metabolism and the Immune Response (B. Fleischer, Ed.) RGLandes, Biomedical Publishers. Georgetown. 1-35 (1995)]. All data are expressed as mean ± standard error.

MALDI-TOF analysis of GIP _1-12 (b) and GLP-1 _7-36 (C) hydrolysis by DPIV and its inhibition by isoleucyl thiazolidide (a) HPLC analysis of serum containing GLP _7-36 metabolites in the presence or absence of the DPIV inhibitor isoleucyl thiazolidide in vivo Effects of DPIV inhibitor isoleucyl thiazolidide on various blood parameters in rats stimulated with duodenal glucose

Claims (4)

  Effector of dipeptidyl peptidase (DPIV) or DPIV-like enzyme activity to lower hyperglycemia levels as found in mammals with clinically inappropriate basal and post-prandeial hyperglycemia Use of.   Administering an effector of dipeptidyl peptidase (DPIV) or DPIV-like enzyme activity to prevent or alleviate pathological metabolic abnormalities in mammals such as diabetes, hyperlipidemia, metabolic acidosis or diabetes mellitus The use of claim 1.   An effector is an inhibitor, a substrate, a pseudo-substrate, a DPIV gene expression inhibitor, a dipeptidyl peptidase (DPIV) or DPIV-like enzyme activity effector containing a binding protein, an anti-dipeptidyl peptidase or an anti-DPIV-like enzyme active antibody, or Use according to claim 1, comprising substances acting as specified effector combinations. An effector of dipeptidyl peptidase (DPIV) or DPIV-like enzyme activity for use in the process of lowering hyperglycemia levels as found in mammals presenting clinically inappropriate postprandial hyperglycemia.